Intracellular chaperones not only regulate protein folding but are also key mediators of the degradation and disposal of misfolded and damaged proteins. There are, however, fundamental gaps in the understanding of how chaperoned proteins are degraded. The novel ubiquitin ligase CHIP binds to the key cytosolic chaperones Hsp70/Hsc70 and Hsp90 and ubiquitinates chaperone-bound client proteins, promoting their proteasomal degradation. The long-term goals of studying CHIP are to elucidate the mechanistic details of CHIP-mediated ubiquitination and to understand how CHIP-mediated ubiquitination affects cellular homeostasis. The objective of this particular application is to define the molecular specificity of the interactions between CHIP and three of its partners: Hsc70, E2 enzymes, and Bag2, an important negative regulator of CHIP. The rationale for the proposed studies is that defining these interactions will establish the architecture of the Hsc70:CHIP ubiquitination complex and explain how this architecture specifies and regulates CHIP- mediated ubiquitination. CHIP-mediated ubiquitination is a crucial part of the life cycle of many proteins involved in diverse pathologies including cancer, cardiovascular diseases and neurodegenerative disorders; CHIP is thus a target of opportunity for pharmacological regulation of such disease-related proteins. The proposed research is relevant to the mission of the NIH in that contributes to the development of fundamental biochemical knowledge that will potentially help to ameliorate diseases that increasingly affect an aging American population. Based on strong preliminary data, three specific aims will be pursued: 1) Identification of specificity determinants that allow CHIP to interact with a limited set of E2 enzymes while excluding others; 2) Elucidation of the mechanism by which Bag2 inhibits CHIP and protects chaperoned proteins from degradation; 3) Determination of how CHIP accesses the substrate binding site of Hsc70 and promotes efficient ubiquitin transfer to chaperone-bound proteins. In the first aim, structure-based mutagenesis and protein-protein interaction affinity measurements will be used to define CHIP:E2 specificity determinants. In the second aim, in vitro ubiquitination assays, NMR and analytical ultracentrifugation will be used to investigate the interactions between domains of Bag2 and their binding sites on Hsc70 and on CHIP. X-ray crystallography will be used to determine the structures of the Bag2 domains alone and bound to their partners. In the third aim, a similar combination of methods will be used to investigate the role of the flexible Hsc70 tail in CHIP-mediated ubiquitination and to determine the structural basis of interactions between CHIP and the Hsc70 substrate-binding domain. These studies are significant because they will elucidate the physical framework of CHIP-mediated ubiquitination of chaperone clients, establish a basis for evaluating whether and how CHIP-mediated ubiquitination is a viable target in the treatment of disease, and yield data that will aid the development of inhibitors for CHIP and other ubiquitin ligases. The proposed studies will characterize the mechanism of CHIP, an important regulator of the degradation of misfolded and damaged proteins in cells. CHIP acts on numerous substrates involved in pathologies such as cancer, cardiovascular disease and neurodegenerative disorders. Therfore, understanding how CHIP carries out its function is expected to apply directly to the understanding of mechanisms underlying such diseases and, potentially, to the treatment of these diseases.